Alzheimer's Disease (AD) has no effective treatments, and recent clinical trials focused on prevention of amy- loid beta (Ab) production have consistently failed. Alternative approaches are urgently needed. We demon- strated that partial inhibition of mitochondrial complex I with small molecule CP2 induced neuroprotection in multiple mouse models of familial AD. While inhibition of complex I activity has been linked to prolonged lon- gevity, we were the first to show that this approach could be beneficial for AD. CP2 penetrates the blood brain barrier, and accumulates in mitochondria where it competes with the flavin mononucleotide for binding to the redox center of complex I. Partial inhibition of complex I activity induced beneficial cellular adaptation to oxida- tive stress and enhancement of cellular energetics, which was associated with a significant delay in the devel- opment of cognitive and behavior phenotypes when independent groups of AD mice were treated at pre- or symptomatic stages of the disease. However, along with the benefits of treatment that included a reduction of soluble and insoluble Ab and pTau, inhibition of glycogen synthase kinase 3b activity, restoration of axonal traf- ficking and levels of brain-derived neurotrophic factor and synaptic proteins, CP2 induced activation of AMP activated protein kinase (AMPK). Taken into consideration detrimental effects reported in relation to the AMPK activation in humans, to achieve efficacious and safe therapeutic response using mitochondrial complex I in- hibitors in the context of AD, it is imperative to understand the molecular mechanisms to avoid failure in trans- lating our findings into clinical settings. The specific experimental goals are: 1) to test the central hypothesis that activation of AMPK is not required for CP2-induced neuroprotection; 2) to determine the hierarchy of mo- lecular mechanisms other than AMPK activation involved in neuroprotection; 3) to confirm these mechanisms in mouse models of AD that express both human A? and Tau protein (3xTgAD) and ApoE4 knock in mice that resemble sporadic AD; 4) to establish blood-based biomarkers for target engagement for translational studies; 5) to determine translational potential of this approach in human neuronal cells; and 6) to conduct phar- macogenomics study using lymphoblastoid cell lines from diverse healthy population to establish safety mar- gins and individualized response to treatment. The proposed studies will provide a rigorous evaluation of molecular mechanisms essential for CP2 neuropro- tection and will investigate the translational potential and safety of this approach for AD using targeted genetic perturbations and advanced techniques in multiple mouse and human model systems. The outcomes will justi- fy the development of combination therapy to mitigate potentially harmful side effects, and biomarkers to moni- tor target engagement and safety. Since the majority of experiments will be conducted in parallel with metfor- min, the only complex I inhibitor that is FDA approved to treat diabetes, the outcomes will also provide the criti- cal biological evidence to establish the rationale and the preclinical criteria, to support further clinical develop- ment of specific MCI inhibitors for AD treatment. ! ! !